Solid compositions for selective adsorption from complex mixtures

ABSTRACT

The present invention relates to a solid and method useful in separating chemical components in a complex mixture when at least one of the chemical components of the mixture is capable of being selectively adsorbed. The solid of the present invention comprises an inorganic substance and moieties (R 10 ) located on at least one surface of the inorganic substance, wherein said inorganic substance is an inorganic oxide and the surface moiety is selected from the group consisting of —CH 2 OH, —CH(OH) 2 , —CH(OH)CH 3 , —CH 2 CH 2 OH, —C(OH) 2 CH 3 , —CH 2 CH(OH) 2  and —CH(OH)CH 2 (OH). Binding moiety, optionally attached to the inorganic substance via a linker, can also be located on the surface of the solid.

CROSS REFERENCE TO RELATED CASES

This is a divisional application of Ser. No. 10/892,779 filed Jul. 16,2004, which is a divisional application of Ser. No. 09/929,621, filedAug. 14, 2001.

FIELD OF THE INVENTION

The present invention concerns solid compositions and methods useful inseparating chemical components in a complex mixture in which at leastone of the chemical components is capable of being selectively adsorbed.The invention also concerns methods of reducing non-specific binding ofthe other components in the mixture.

BACKGROUND OF THE INVENTION

Separations of components in a mixture have been important in manyscientific disciplines, such as chemistry, biochemistry and molecularbiology. The separation of components in a mixture allows isolation ofthe component of interest, i.e., an analyte. After the analyte isisolated, the properties of the analyte can be studied or used. Withoutthe separation, it may be difficult to determine the properties of theanalyte because, whatever the measurement technique used, the propertiesof the component of interest could be masked or influenced by othercomponents in the mixture. Thus, separation techniques can be consideredas corner stones in scientific studies.

The separation of components is made difficult if the mixture containingthe analyte is a complex mixture. Good examples of complex mixturesinclude media of biological fermentation, cell cultures, transgenicallyproduced milk, or slurries of transgenic plant matter, in which aspecific analyte is desired and needs to be separated and purified. Theseparation of components in the complex mixture is usually accomplishedby affinity separation techniques. The affinity separation techniqueusually involves contacting the mixture with a solid phase having afunctionality specifically designed to bind to the analyte, but besubstantially non-reactive with other components in the mixture, therebyleaving the other components free to be removed. After the non-boundcomponents are removed, e.g. by washing the solid phase with water orbuffer, the analyte is left behind bound to the solid phase, so theanalyte is separated from the non-bound components. The analyte is thenisolated by separating the analyte from the solid phase, usually by abuffer change, to recover the analyte as free molecules.

Classes of valuable “affinity” techniques for purification have beendeveloped. These techniques have many names, affinity chromatography,affinity precipitation, immunoaffinity separation, etc., but they allrely on the same principles, that is, a specific functionality orbinding moiety is chemically attached to a solid support that binds veryselectively to the target analyte. The most common binding moieties forprotein purification are other proteins such as Protein A or Protein G,or monoclonal antibodies, chelated metals ions, polypeptides, or smallorganic molecules. Monoclonal antibodies can be especially attractivefor protein purification because they can be highly selective for thetarget protein. As indicated above, the mixture that contains theanalyte is allowed to contact the affinity solid support with thebinding moiety attached. The analyte binds to the binding moiety on thesupport and the rest of the mixture is removed. The analyte is thenremoved from the binding moiety by elution, usually achieved by changingthe solvent. Very high purification factors can be realized. There isextensive literature on affinity techniques¹⁻⁸.

Recent developments in the selection and production of monoclonalantibodies have made the affinity technique based on the monoclonalantibody as the binding moiety a very powerful technique for thepurification of proteins and biopharmaceuticals. Monoclonal antibodiesare proteins themselves that are often purified from cell culture orfermentation using affinity purification that uses Protein A or ProteinG as the ligand. New small organic Protein A mimetics have also beendescribed as useful ligands for monclonal antibody purification.

Although affinity purification has proven to be a powerful technique,its full potential has not been fully realized. It is most commonlypracticed where the support is formed into small beads, on the order of0.05 to 0.5 mm or so, and the beads, often referred to as media, areloaded into a chromatography column. The mixture to be purified is thenpassed through the column and the analyte binds to the binding moietyattached to the media. The column is then washed extensively to removethe occluded mixture. An elution solvent is then passed through thecolumn liberating the analyte in solution. On a large-scale, thisprocess requires that the media have good physical strength to handlethe weight and turbulence encountered in column applications.

Certain supports currently used in affinity separations, whether ascolumn chromatography or some other system, are low surface areamaterials, such as carbohydrate-based materials or polymers. These lowsurface area supports can have low capacity. Because of the lowcapacity, relatively large loadings of media are needed to recover thetarget species. But, with large loadings of media, flow rates over thecolumn are restricted to low rates due to pressure drop considerations.Column chromatography can also be practiced under high pressure wheresmaller beads are used to increase the capacity of the media. Becausethese beads must have higher strength to handle the pressure,carbohydrate gels are cross-linked, thereby lowering the capacity of theresulting beads. Therefore, there is a need to provide affinity supportswith high capacity and which are further physically robust when used inhigh pressure liquid chromatography.

Developing high surface area supports is one approach to obtaining highcapacity affinity separation media. With a higher capacity material,smaller amounts of the affinity support is needed to recover the targetspecies, column pressure drops are lower, flow rates are higher, andthere is less occluded feed contamination. High surface areas couldrange from 10-500 m²/g. Materials that can provide high surface area aresilica gels, silicas, aluminas, zirconias, carbohydrates, and polymericmaterials such as macropore acrylic beads. In the case of silica gels,surface areas can vary from very low, 1 m²/g, to very high, in excess of800 m²/g, with pore size modes from very low, less that 25 Å to inexcess of 1500 Å. Furthermore, inorganic oxide-based materials areusually much more physically robust than the softer carbohydrate basedsupports.

When used as media in affinity separation techniques with a bindingmoiety attached, these oxide based materials, while having the requisitehigh surface area, can suffer from a high degree of non-selectivebinding of unwanted materials. Not all of the surface area will be usedfor the affinity separation; some will actually provide surface regionsfor non-selective adsorption. It is well known that proteins bind verystrongly to silica for instance, sometimes irreversibly andnon-selectively. Therefore, while the binding moiety can be veryselective, the unused regions of the surface will be non-selective. Thenet effect is to lower the selectivity of the high surface areamaterials, thereby reducing the purification factors of the overallprocess. This non-selective adsorption by many oxide supports, andespecially silicas such as silica gels, is the reason these materialsare currently not used extensively as affinity separation supports.

One of the objectives of this invention to describe a surfacecomposition to be applied to high surface area materials which improvethe non-selective adsorption while retaining the high capacity for theselective affinity binding.

Such compositions will have great value in “affinity separations” fromcomplex biological mixtures where specific biological species, such asproteins, are synthesized by genetically engineered organisms. Forinstance the complex mixture might be a fermentation broth for cellularor bacterial production of a target protein. The fermentation broths arecomplex mixtures of proteins, carbohydrates, etc., that support theorganism growth, as well as by products produced by the fermentation.The target species can also be produced from the fermentation and isproduced by the organism into the broth. In some cases, the targetspecies is produced in the cell. Recovery is therefore complicated bythe fact the cells need to be homogenized and the target dissolved.These mixtures are particularly insidious for target species isolationand purification. Separation and purification schemes for the isolationand purification of the target species from fermentation broths are verycomplicated and expensive. The cost of isolation and purification isespecially significant as the large-scale production. Because of thechallenging nature of this problem, the field of purification andisolation is extensive.

SUMMARY OF THE INVENTION

The solid composition of the invention comprises an inorganic substanceand moiety R₁₀ located on at least one surface of said inorganicsubstance, wherein

-   -   said inorganic substance is an inorganic oxide    -   and said R₁₀ group is an entity selected from the group        consisting of —CH₂OH, —CH(OH)₂, —CH(OH)CH₃, —CH₂CH₂OH,        —C(OH)₂CH₃, —CH₂CH(OH)₂ and —CH(OH)CH₂(OH).

When R₁₀ is an entity selected from the group consisting of —CH₂OH,—CH(OH)₂, —CH(OH)CH₃, —CH₂CH₂OH, —C(OH)₂CH₃, —CH₂CH(OH)₂ and—CH(OH)CH₂(OH), the solid support possesses a distinctive characteristicof having reduced non-specific binding of any non-analyte components ina complex mixture. The members of R₁₀ have a common property of havingzero electric charge and being hydrophilic. Without being held to anyparticular theory, it is believed that when the solid support has any ofthe R₁₀ entities, i.e.—CH₂OH, —CH(OH)₂, —CH(OH)CH₃, —CH₂CH₂OH,—C(OH)₂CH₃, —CH₂CH(OH)₂ and —CH(OH)CH₂(OH), located on its surface, thebinding of the non-analyte component in the mixture to the surface hasan entropy change lower than the remaining non-analyte component in themixture's aqueous phase. Binding of any component, e.g., non-analyte oranalyte component, from a solution to a surface involves a lowering ofentropy due to localization of the non-analyte on the surface. In orderfor binding to be favorable, there has to be an interaction betweennon-analyte and surface, such as a Columbic charge interaction orhydrophobic interaction, to overcome the lowering of entropy due tosurface localization. Coating with any one of or any mixture of theentities for R₁₀, however, produces a hydrophilic surface, as well as asurface having a zero net charge which reduces interaction necessary forreducing entropy and accordingly reduces non-binding of non-analyte.

Also within the scope of the present invention are solids comprising theinorganic substance, moiety R₁₀ located on at least one surface of theinorganic substance and at least one binding moiety capable of bindinganalyte. The moiety R₁₀ is preferably located on a portion of theinorganic oxide's surface and on the remainder of the surface is locatedbinding moiety. The binding moiety is selected to provide bonding to aparticular analyte and provides the highly selective binding foraffinity separations, while the surface coated with R₁₀ groups reducesnon-selective binding of other species. In preferred embodiments, asignificant, if not a large majority, of the inorganic oxide surface iscovered by R₁₀ groups, e.g., at concentrations of about 1 to 10 groupsper nm² substrate, and the remainder covered by the binding moiety.

Also within the scope of the invention is a solid comprising theinorganic substance in which binding moiety is optionally located on theinorganic oxide surface through a linker. The linker can be located onthe inorganic oxide surface by reacting a linker compound with theinorganic oxide and then subsequently reacting the linker with thebinding moiety. Accordingly, the invention contemplates inorganicsubstances comprising linker groups and R₁₀ moieties located on itssurface. This solid in turn can be transferred to an end user who canattach a specifically designed binding moiety to the linker for use in aseparation.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 schematically depicts the composition of this inventioncomprising the inorganic substance, R₁₀ surface moieties, the optionallinker, binding moiety and analyte.

FIG. 2 shows the results of Examples 1 and 2, with lanes 2 and 7representing Pharmacia 3.6-9.3 Broad pI Standard, lanes 3 and 4representing Example 1 and lanes 5 and 6 representing Example 2. Thisfigure illustrates non-specific binding of untreated conventionalinorganic oxides.

FIG. 3 shows the results of Examples 3-5.

FIG. 4 shows the results of Examples 6-8 and is illustrative of theinvention.

FIGS. 5-8 contain results from analyses conducted to characterize aparticular embodiment of the invention (Example 8), with FIG. 5 showingthe diffuse reflectance IR spectrum of Example 8, FIG. 6 showing thesame spectrum for the composition prepared in Example 7 for comparison,FIG. 7 showing the MAS Si²⁹ NMR spectrum of Example 8, and FIG. 8showing the X-ray photoelectron spectrum, XPS, of Example 8.

FIG. 9 shows the XPS spectrum of Example 7.

FIG. 10 shows the chromatogram of the loading, wash and elution from acolumn prepared in Example 9 using the invention after attachment ofbinding moiety.

FIG. 11 depicts the absorbance at 280 nm of the effluent from the sizeexclusion chromatography of purified rabbit polyclonal IgG obtained fromthe eluent of the affinity chromatography conducted in Example 9.

FIG. 12 depicts the absorbance at 280 nm of the effluent from the sizeexclusion chromatography of the starting rabbit polyclonal IgG that wasspiked into a cell broth in Example 9.

FIG. 13 depicts the preparation of a coating agent that yields —CH₂OH asR₁₀, from reaction illustrated in FIG. 14.

FIG. 14 shows the preparation of silica having R₁₀ attached via asilicon atom which is not a part of the silica, in which R₁₀ is —CH₂OH,so that —Si—CH₂OH is directly attached to the silica (HO—Si—represents asilanol group on the surface of silica).

FIG. 15 depicts the preparation of a coating agent that yields —CH(OH)₂as R₁₀, from reaction illustrated in FIG. 16.

FIG. 16 shows the preparation of silica having R₁₀ indirectly attachedvia a silicon atom which is not a part of the silica, so that —Si—R₁₀groups are attached to the silica's surface, in which R₁₀ is —CH(OH)₂(HO—Si—represents a silanol group on the surface of silica).

FIG. 17 shows the preparation of a coating agent that yieldshydroxyethyl as R₁₀, from reaction illustrated in FIG. 18.

FIG. 18 shows a method for the preparation of a solid comprising silicaand —Si—R₁₀ groups attached to the surface of the silica, i.e. R₁₀ isindirectly attached via a silicon atom which is not a part of the silicato the silica surface, wherein R₁₀ is 1,2-dihydroxyethyl.

FIG. 19 shows another method for preparing a solid comprising silica and—Si—R₁₀ groups attached to the surface of the silica, i.e. R₁₀ isindirectly attached via a silicon atom which is not a part of the silicato the silica surface, wherein R₁₀ is 1,2-dihydroxyethyl.

FIG. 20 shows an embodiment of the present invention in which —Si—R₁₀groups are crosslinked when attached to the surface of silica, whereinR₁₀ is hydroxymethyl (HO—Si—represents a silanol group on the surface ofsilica).

FIG. 21 shows the preparation of a coating agent that would lead to the—Si—R₁₀ group attaching to the surface of silica at a single point,wherein R₁₀ is hydroxymethyl, resulting from the reaction illustrated inFIG. 22.

FIG. 22 depicts an embodiment of the present invention in which the—Si—R₁₀ group is attached to the surface of silica at a single point,wherein R₁₀ is hydroxymethyl (HO—Si—represents a silanol group on thesurface of silica).

DETAILED DESCRIPTION OF THE INVENTION

Inorganic Substance

Inorganic substances suitable for making the invention include thoseproducts commercially available as chromatographic media. Thesesubstances can be prepared using methods known in the art. The inorganicsubstance can also be considered a support for the binding moiety laterdescribed below and from time to time the inorganic substance isreferred to herein as a “support.” In general, the inorganic substanceof the present invention is an inorganic oxide, more suitably aninorganic metal oxide, silicate or aluminosilicate. Inorganic metaloxide is preferred. Inorganic oxides suitable for this invention havefree hydroxyl groups capable of bonding to or reacting with otherchemical functionalities. It is through those hydroxyl groups that R₁₀surface moieties and binding moieties and/or linkers are reacted orbonded. In general, suitable inorganic oxides include those having about1 to about 10 hydroxyl groups per square nanometer of solid inorganicoxide.

Examples of the preferred inorganic metal oxide include silica such aschromatographic grade silica or silica gel, alumina, silica-alumina,zirconia, zirconate, controlled pore glass or titania. The inorganicmetal oxide preferably is silica, more preferably chromatographic gradesilica or silica gel. Magnetically responsive inorganic metal oxides,such as siliceous oxide-coated magnetic particles disclosed in WO98/31461 (the disclosure of which is incorporated by reference) are alsosuitable. Mixed inorganic metal oxides, e.g. cogels of silica andalumina, or coprecipitates can also be used. Solids prepared from sodiumsilicate are examples of a suitable silicate and zeolite is an exampleof a suitable aluminaosilicate. The solid of the present invention canbe in a physical form of particulates, fibers and plates.

Surface Moieties (R₁₀)

As indicated earlier, R₁₀ groups are selected from the group consistingof —CH₂OH, —CH(OH)₂, —CH(OH)CH₃, —CH₂CH₂OH, —C(OH)₂CH₃, —CH₂CH(OH)₂ and—CH(OH)CH₂(OH). R₁₀ preferably is an entity selected from the group of—CH₂OH, —CH(OH)CH₃, —CH₂CH₂OH, and —CH(OH)CH₂(OH). More preferably, R₁₀is an entity selected from the group of —CH₂OH, —CH(OH)CH₃ and—CH₂CH₂OH. Most preferably R₁₀ is —CH₂OH.

The moiety R₁₀ is located on at least one surface of the inorganicsubstance. By “located” it is meant R₁₀ can be attached directly to afunctionality on the surface of the starting inorganic substance. R₁₀can be located on surface area present on the periphery of the inorganicsubstance, or located on surface area presented in pores which penetrateinto the interior of the inorganic substance and have (pore) openings onthe substance's periphery.

R₁₀ can also be “located” on the surface of the inorganic substance bybeing attached to the inorganic substance surface via bivalent moiety oratom (—X—) to form a group having the formula —X—R₁₀. The bivalentmoiety or atom linking R₁₀ in this embodiment is not present in thecomposition of the starting inorganic substance prior to reaction of thesubstance with the reactant. The moiety or atom can be from a reactantemployed to create R₁₀, e.g., a residual metal atom (e.g. silicon atom),originating from a silane reactant. The residual moiety or atom isattached directly to said inorganic substance, and preferably throughhydroxyl groups on the surface of the inorganic substances. The —X—group in such reactants vary from reactant to reactant, but can be metalatoms or other chemical moieties. For example, X can be derived frommetal atoms such as silicon, aluminum, zirconium or the like. Theinorganic substance selected may also determine the selection of —X— andits associated reactant. Generally, any reactant containing —X— will bethat which can react with reactive functionality on the inorganicsubstance. In the case of inorganic oxides, suitable reactants typicallyare those capable of reacting with hydroxyl groups.

The chemistry of reacting compounds, e.g., those capable of creatingR₁₀, with the inorganic substances is known in the art, e.g., Smith,Organic Synthesis, John Wiley & Sons, 1994; March, Advanced OrganicChemistry, John Wiley & Sons, Fourth Edition, 1992; Larock,Comprehensive Organic Transformations, John Wiley & Sons, SecondEdition, 1999; Greene et al, Protective Groups in Organic Synthesis,John Wiley & Sons, Third Edition, 1999; Brook, Silicon in Organic,Organometallic, and Polymer Chemistry, John Wiley & Sons, 2000;Hermanson et al, Immobilized Affinity Ligand Techniques, 1992; Weetall,“Covalent Coupling Methods for Inorganic Support Materials”, in Methodsin Enzymology, vol. XLIV, edited by K. Mosbach, pp. 134-148, 1976;Abbott, U.S. Pat. No. 4,298,500; and Arkles, U.S. Pat. No. 5,371,262;the disclosures of these documents are herein incorporated by reference.For example, a solid comprising R₁₀ groups located on the inorganicsubstance's surface can be prepared from a reactant or coating agentsuch as alkoxysilane, dialkoxysilane or trialkoxysilane bearing aprecursor group of R₁₀. For instance, acetoxymethyl can be the precursorgroup of hydroxymethyl. The coating agent is then allowed to react withthe surface of the inorganic substance, followed by hydrolysis of theprecursor to produce an inorganic substance having R₁₀ groups attached.

A method for preparing silica having —CH₂OH as R₁₀ located on the silicasurface is shown in FIGS. 13 and 14. FIG. 13 depicts the preparation ofa coating agent, acetoxymethyltriethoxysilane (see Compound (2)), forintroducing Si—R₁₀ groups to silanol groups at the surface of silica,i.e. HO—Si—, in which R₁₀ is hydroxymethyl (see the reactions presentedin FIG. 14, in which Compound (5) is silica having Si—R₁₀ directlyattached at the surface wherein R₁₀ is hydroxymethyl). In other words,in FIG. 14, a method is shown for introducing R₁₀ groups to the surfaceof silica via a Si atom which is an example of the residual moiety oratom X described above as being residual from the reactant and which isnot a part of the starting inorganic substance.

A method for the preparing silica comprising R₁₀, wherein R₁₀ is—CH(OH)₂ is shown in FIGS. 15 and 16. FIG. 15 depicts the preparation ofa coating agent, diacetoxymethyltriethoxysilane (see Compound (7)), forintroducing —CH(OH)₂ groups as the R₁₀ group to the surface of silica(see the reactions and Compound (9) presented in FIG. 16).

FIG. 17 shows a method for preparing a coating agent,acetoxyethyltriethoxysilane (see Compound (11)), for introducing2-hydroxyethyl to the surface of silica.

Two methods for the preparation of a solid comprising silica and1,2-dihydroxyethyl as R₁₀ groups attached to the surface of the silicaare depicted in FIGS. 18 and 19.

Also, within the scope of the present invention are solids comprisingthe inorganic substance having R₁₀ groups attached to a surface of thesolid via a residual metal (e.g., Si) from the silane reactant whereineach resulting Si—R₁₀ group is attached to the inorganic substance viathree covalent bonds (e.g. see the final products of the reactionschemes in FIGS. 14, 16, 18 and 19, resulting from the reaction of acoating agent having three silanol groups).

As seen in FIGS. 21 and 22, it is also believed coating agents can beselected so that residual atoms can also be attached to the inorganicsubstance via one or two covalent bonds, or that certain embodimentscomprise crosslinking of the Si atoms. Such crosslinking can be aSi—O—Si linkage or another linkage such as Si—O—C(O)—O—Si,Si—O-alkylene-O—Si or Si—O—C(O)-alkylene-O—Si). The final product, i.e.Compound (20), of the reaction scheme in FIG. 22 illustrates anembodiment of the solid of the present invention in which Si—R₁₀ grouphas a single point of attachment to the surface of the silica. Thatembodiment is prepared from a reaction of the solid inorganic substanceand mono-ethoxysilane (see FIG. 21 for the preparation of the coatingagent which is a mono-ethoxysilane).

Binding Moiety

The solid of the present invention can further comprise at least onebinding moiety which is attached to, optionally via a linker, orotherwise located on the surface of said inorganic substance. Thebinding moiety is any molecule or molecule fragment capable of bindingto another moiety or molecule-based analyte, e.g., binding throughhydrophobic interaction, covalent bonding or Columbic interaction. Suchmoieties are well known to those skilled in the separations industry.Moieties typically used in the bioseparations industry include (e.g.biotin, avidin and streptavidin), natural or unnatural protein, peptide,antigen and nucleic acid. As the binding moiety of the solid of thepresent invention, the protein is preferably a receptor or antibody.

It is also preferred that, in the solid of the present invention, thebinding moiety is ligand, a receptor, antibody, antigen, DNA or RNA,including hybridization probes for nucleic acids. When the ligand isavidin or streptavidin, the analyte can be biotin or biotinylated, andvice versa.

The binding moiety is attached to the inorganic substance using methodsknown in the art (e.g. Hermanson et al, Immobilized Affinity LigandTechniques, Academic Press, 1992 and the other references cited earlierwith respect to attaching R₁₀ moieties). In solids comprising inorganicoxides, the binding moiety can be attached via a reaction with surfacefunctional groups, e.g., hydroxyl, on the starting inorganic oxide.

Alternatively, the binding moiety can be attached to the inorganicsubstance via a linker. The linker can be a bivalent chemical group,which is optionally substituted. The optionally substituted bivalentchemical group can comprise n —R— groups, with n being the number of —R—groups, n being an integer of at least 1, preferably not larger than 30,and more preferably not higher than 15. More typically, the bivalentchemical group is about 1 to about 30 atoms, preferably about 1 to about20 atoms, more preferably about 5 to about 15 atoms, in length measuredfrom the binding moiety to the inorganic substance. The chemical group—R— can be selected from the group consisting of —C(R₁)H—, —C(R₂)═C(R₃)—and —C≡C—, where R₁, R₂ and R₃ independently being H, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, aralkylor substituted aralkyl, said —R— group optionally replaced with —O—,—S—, carbonyl, thiocarbonyl, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—,—OC(S)—, —C(S)O—, —C(S)S—, —SC(S)—, —N(R₄)—, —N(R₄)C(O)—, —C(O)N(R₄)—,—C(R₅)═N—, —N═C(R₅)—, —C(R₅)═NO—, —ON═C(R₅)—, —P—, —P(OH)O—, arylene,substituted arylene, cycloalkylene, substituted cycloalkylene,cycloalkenylene, substituted cycloalkenylene, bivalent heterocyclyl orbivalent substituted heterocyclyl, where R₄ and R₅ independently beingH, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl,alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl,aryl, substituted aryl, aralkyl or substituted aralkyl. Illustrative ofthe chemical group is “hydrocarbyl” comprising n —R— groups and whereinn is described above, at least one —R— group is —CH₂— and (n−1)—R—groups are optionally replaced with the R groups mentioned above, e.g.,—O—, —S—, etc.

“Substituted” is used herein to mean containing pendent substituentgroups that do not alter the predominant chemical character of thesubstituted R group, e.g., hydrocarbon character for hydrocarbyls.

The term “alkyl” refers to a saturated branched or unbranchedhydrocarbyl radical, preferably those of 1 to 30, more preferably 1 to20 and even more preferably 1 to 6, carbon atoms. Examples of “alkyl”include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, isopentyl,neopentyl, 1,1-dimethylpropyl, n-hexyl, 1-methylpentyl, 2-methylpentyl,3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, isohexyl andneohexyl. The term “cycloalkyl” refers to a saturated cyclic hydrocarbylradical, preferably of 3 to 10, and more preferably 3 to 6, carbonatoms. Examples of “cycloalkyl” include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, bicycloheptyl and decalin. “Alkenyl” is abranched or unbranched hydrocarbyl radical having at least one C═C bond,wherein the hydrocarbyl radical is preferably of 2 to 30, morepreferably 2 to 20 and even more preferably 2 to 6, carbon atoms.Examples of “alkenyl” include vinyl, allyl, 1-propenyl, isopropenyl,2-butenyl, 1,3-butadienyl, 3-pentenyl and 2-hexenyl. “Cycloalkenyl”refers to a cyclic hydrocarbyl radical, preferably of 3 to 10,preferably 3 to 6, carbon atoms having at least one C═C bond. “Alkynyl”is a branched or unbranched hydrocarbyl radical, preferably of 2 to 30,more preferably 2 to 20 and even more preferably 2 to 6, carbon atomshaving at least one —C≡C— bond. Examples of “alkynyl” include ethynyl,1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl and 2-penten-4-ynyl.“Cycloalkynyl” is a cyclic hydrocarbyl radical preferably of 3 to 10,more preferably 3 to 6, carbon atoms having at least one —C≡C— bond.Examples of “cycloalkynyl” include pentynyl and hexynyl. “Aryl” is anaromatic cyclic hydrocarbyl radical, preferably of 6 to 14 carbon atoms.Examples of “aryl” include phenyl, naphthyl, anthracyl and phenanthryl,with phenyl being the preferred aryl. “Aralkyl” is an alkyl radicalsubstituted with one or more aryl radical.

Examples of “aralkyl” include benzyl, phenethyl, diphenylmethy andtrityl, with benzyl being the preferred aralkyl. “Bivalent heterocyclyl”are bivalent cyclic radicals typically having 3 to 10, preferably 3 to7, more preferably 4 to 6, ring atoms with 1 to 4 of the ring atomsbeing O, S or N atoms, or mixture of O, S and/or N atoms. Examples ofbivalent heterocyclyl include bivalent radicals of thiirene, oxirane,aziridine, 1H-azirine, 2H-azirine, 2H-thiete, thietane, 2H-oxete,oxetane, azete, azetidine, 1,2-oxazetidine, thiophene, furan, pyrrole,imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole,1,3-dioxolane, 1,2,3-thiadiazole, 1,3,4-thiadiazole, 1,2,4-thiadiazole,1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, 1,2,5-oxadiazole,1,2,3-triazole, 1,2,4-triazole, tetrazole, oxadiazole, pyridine,quinoline, isoquinoline, quinolizine, quinazoline, pteridine, carbazole,benzoxazole, 1,3-oxazine, 2H-1,3-oxazine, phenazine, phenothiazine,pyridazine, pyrimidine, pyrazine, benzo[b]furan, benzo[b]thiophene,indole, isoindole, indazole, purine, isobenzofuran, tetrahydrofuran,1,4-dioxane, pyrrolidine, tetrahydropyran, 1,2-dihydropyridine,1,4-dihydropyridine, piperidine, piperazine, morpholine, thiomorpholine,chroman, isochroman, chromene, 1H-azepine, 3H-azepine, 1,2-diazepine,1,3-diazepine, 1,4-diazepine, triazepines and azocine. “Heteroaryl”refers to aromatic heterocyclic radicals. “Alkylene”, “alkenylene”,“alkynylene”, “cycloalkylene”, “cyclalkenylene” and “arylene” arebivalent equivalents of the alky, alkenyl, alkynyl, cycloalkyl,cycloalkenyl and aryl radicals, respectively.

“Substituted alkyl” is an alkyl substituted with 1 to 5, preferably 1 to3, substituents selected from the group consisting of hydroxy,sulfydryl, alkoxy, alkylthio, amino, alkylamino, dialkylamino,arylamino, N,N-arylalkylamino, diarylamino, azido, amidino, ureido,fluoro, chloro, bromo, iodo, nitro, cyano, acyl (preferably acetyl andbenzoyl), thioacyl, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido,alkylsulfamoyl, carboxyl, alkylcarbonyloxy (preferably acetoxy),arylcarbonyloxy (preferably benzoyloxy), alkoxycarbonyloxy,aryloxycarbonyloxy, carbamoyl, aryl (preferably phenyl), styryl,cycloalkyl, cycloalkenyl and heterocyclyl (preferably heteroaryl).

“Substituted alkenyl” is an alkenyl substituted with 1 to 5, preferably1 to 3, substituents selected from the group consisting of hydroxy,sulfydryl, alkoxy, alkylthio, amino, alkylamino, dialkylamino,arylamino, N,N-arylalkylamino, diarylamino, azido, amidino, ureido,fluoro, chloro, bromo, iodo, nitro, cyano, acyl (preferably acetyl andbenzoyl), thioacyl, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido,alkylsulfamoyl, carboxyl, alkylcarbonyloxy (preferably acetoxy),arylcarbonyloxy (preferably benzoyloxy), alkoxycarbonyloxy,aryloxycarbonyloxy, carbamoyl, aryl (preferably phenyl), styryl,cycloalkyl, cycloalkenyl and heterocyclyl (preferably heteroaryl).

“Substituted alkynyl” is an alkynyl substituted with 1 to 5, preferably1 to 3, substituents selected from the group consisting of hydroxy,sulfydryl, alkoxy, alkylthio, amino, alkylamino, dialkylamino,arylamino, N,N-arylalkylamino, diarylamino, azido, amidino, ureido,fluoro, chloro, bromo, iodo, nitro, cyano, acyl (preferably acetyl andbenzoyl), thioacyl, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido,alkylsulfamoyl, carboxyl, alkylcarbonyloxy (preferably acetoxy),arylcarbonyloxy (preferably benzoyloxy), alkoxycarbonyloxy,aryloxycarbonyloxy, carbamoyl, aryl (preferably phenyl), styryl,cycloalkyl, cycloalkenyl and heterocyclyl (preferably heteroaryl).

“Substituted cycloalkyl” is a cycloalkyl substituted with 1 to 5,preferably 1 to 3, substituents selected from the group consisting ofalkyl, alkenyl, alkynyl, aralkyl, hydroxy, sulfydryl, alkoxy, alkylthio,amino, alkylamino, dialkylamino, arylamino, N,N-arylalkylamino,diarylamino, azido, amidino, ureido, fluoro, chloro, bromo, iodo, nitro,cyano, acyl (preferably acetyl and benzoyl), thioacyl, alkylsulfinyl,alkylsulfonyl, alkylsulfonamido, alkylsulfamoyl, carboxyl,alkylcarbonyloxy (preferably acetoxy), arylcarbonyloxy (preferablybenzoyloxy), alkoxycarbonyloxy, aryloxycarbonyloxy, carbamoyl, aryl(preferably phenyl), styryl, cycloalkyl, cycloalkenyl and heterocyclyl(preferably heteroaryl).

“Substituted cycloalkenyl” is a cycloalkenyl substituted with 1 to 5,preferably 1 to 3, substituents selected from the group consisting ofalkyl, alkenyl, alkynyl, aralkyl, hydroxy, sulfydryl, alkoxy, alkylthio,amino, alkylamino, dialkylamino, arylamino, N,N-arylalkylamino,diarylamino, azido, amidino, ureido, fluoro, chloro, bromo, iodo, nitro,cyano, acyl (preferably acetyl and benzoyl), thioacyl, alkylsulfinyl,alkylsulfonyl, alkylsulfonamido, alkylsulfamoyl, carboxyl,alkylcarbonyloxy (preferably acetoxy), arylcarbonyloxy (preferablybenzoyloxy), alkoxycarbonyloxy, aryloxycarbonyloxy, carbamoyl, aryl(preferably phenyl), styryl, cycloalkyl, cycloalkenyl and heterocyclyl(preferably heteroaryl).

“Substituted cycloalkynyl” is a cycloalkynyl substituted with 1 to 5,preferably 1 to 3, substituents selected from the group consisting ofalkyl, alkenyl, alkynyl, aralkyl, hydroxy, sulfydryl, alkoxy, alkylthio,amino, alkylamino, dialkylamino, arylamino, N,N-arylalkylamino,diarylamino, azido, amidino, ureido, fluoro, chloro, bromo, iodo, nitro,cyano, acyl (preferably acetyl and benzoyl), thioacyl, alkylsulfinyl,alkylsulfonyl, alkylsulfonamido, alkylsulfamoyl, carboxyl,alkylcarbonyloxy (preferably acetoxy), arylcarbonyloxy (preferablybenzoyloxy), alkoxycarbonyloxy, aryloxycarbonyloxy, carbamoyl, aryl(preferably phenyl), styryl, cycloalkyl, cycloalkenyl and heterocyclyl(preferably heteroaryl).

“Substituted aryl” is an aryl substituted with 1 to 5, preferably 1 to3, substituents selected from the group consisting of alkyl, alkenyl,alkynyl, aralkyl, hydroxy, sulfydryl, alkoxy, alkylthio, amino,alkylamino, dialkylamino, arylamino, N,N-arylalkylamino, diarylamino,azido, amidino, ureido, fluoro, chloro, bromo, iodo, nitro, cyano, acyl(preferably acetyl and benzoyl), thioacyl, alkylsulfinyl, alkylsulfonyl,alkylsulfonamido, alkylsulfamoyl, carboxyl, alkylcarbonyloxy (preferablyacetoxy), arylcarbonyloxy (preferably benzoyloxy), alkoxycarbonyloxy,aryloxycarbonyloxy, carbamoyl, styryl, cycloalkyl, cycloalkenyl, aryl(preferably phenyl) and heterocyclyl (preferably heteroaryl).

“Substituted heterocyclyl” is a heterocyclyl radical substituted with 1to 5, preferably 1 to 3, substituents selected from the group consistingof alkyl, alkenyl, alkynyl, aralkyl, hydroxy, sulfydryl, alkoxy,alkylthio, amino, alkylamino, dialkylamino, arylamino,N,N-arylalkylamino, diarylamino, azido, amidino, ureido, fluoro, chloro,bromo, iodo, nitro, cyano, acyl (preferably acetyl and benzoyl),thioacyl, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido,alkylsulfamoyl, carboxyl, alkylcarbonyloxy (preferably acetoxy),arylcarbonyloxy (preferably benzoyloxy), alkoxycarbonyloxy,aryloxycarbonyloxy, carbamoyl, aryl (preferably phenyl), styryl,cycloalkyl, cycloalkenyl and heterocyclyl (preferably heteroaryl).

“Substituted arylene”, “substituted cycloalkylene”, “substitutedcycloalkenylene”, “substituted bivalent heterocyclyl” and “substitutedaralkyl” are bivalent equivalents of “substituted aryl”, “substitutedcycloalkyl”, “substituted cycloalkenyl” and “substituted heterocyclyl”.

The linkage connecting the chemical group —R— of the linker andinorganic substance depends on the chemistry employed to react thelinker and inorganic substance. The linkage can be an ether, thioether,ester, thioester, carbonate, carbamate, phosphate, phosphonate,phosphoester, phosphoramidate, amine, amide, imide, urea, thiourea,sulfonamide, sulfoxide, sulfone, disulfide, oxime, O-acyl oxime,O-carbamoyl oxime, O-acyloxyalkyl oxime, O-acyloxyalkyloxy oxime,O-oximinophosphate, O-oximinophosphonate, O-oximinophosphoramidate or—C═C-linkage. The linkage connecting the chemical group —R— and bindingmoiety can also be one of the aforementioned linkages.

The chemistry of reacting linkers to substances (e.g., inorganicsubstances) is well described in the literature (see Hermanson et al,Immobilized Affinity Ligand Techniques, 1992 and Weetall, Methods inEnzymology, vol. XLIV, pp. 134-148, 1976). The particular chemistry forreacting linker to inorganic substances depends on the inorganicsubstance and linker employed. Likewise, the chemistry of reacting thelinker to binding moiety depends on the linker and binding moietyemployed. Specific examples of suitable linker/binding moiety couplingchemistry are shown in Table 1. According to Table 1, the binding moietycan be coupled to the linker via an amino, sulfhydryl, carbonyl orhydroxy group or an active hydrogen atom on the binding moiety. TABLE 1Examples of Conventional Linker/Binding Moiety Coupling ChemistryBinding Moiety Linkers Formed With Coupling Group Cyanogen bromide(CNBr) Amino N-Hydroxy succinimide esters Amino Carbonyl diimidazoleAmino Reductive animation Amino FMP activation* Amino EDC-mediated amidebond formation** Amino Organic sulfonyl chlorides: tosyl chloride Aminoand tresyl chloride Divinylsulfone Amino Azlactone Amino Cyanuricchloride (trichloro-s-triazine) Amino Iodoacetyl or bromoacetylactivation methods Sulfhydryl Maleimide Sulfhydryl Pyridyl disulfideSulfhydryl Divinylsulfone Sulfhydryl Epoxy Sulfhydryl TNB-Thiol***Sulfhydryl Hydrazide Carbonyl Reductive amination Carbonyl Epoxy(bisoxirane) Hydroxy Divinylsulfone Hydroxy Cyanuric chloride HydroxyDiazonium compounds Active hydrogen Mannich condensation Active hydrogen*FMP means 2-fluoro-1-methyl-pyridinium toluene-4-sulfonate**EDC means 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide***TNB-thiol means 2-iminothiolane 5,5-dithio-bis-(2-nitrobenzoic acid)

In making solid supports comprising linker groups, the order of creatinglinker groups in conjunction with adding R₁₀ groups to the inorganicsubstance can vary. The R₁₀ can be created on the inorganic surfaceafter attaching the linker, or it can be created prior to reactinglinker. Alternatively, precursors to either R₁₀ or the linker or bothcan be created and/or attached, with the precursors later reacted tocreate the final R₁₀ and/or linker.

The concentration of linker groups on the inorganic surface can vary. Incertain embodiments of the present invention, the binding moietycomprises large protein molecules which can “shadow” large regions ofthe support's surface area. As a result, the concentration of the linkersites on the support's surface does not need to be relatively high. Theconcentration can be optimized based on the size of the contemplatedbinding moiety/analyte complex.

Factors that determine concentrations of R₁₀ and binding moiety includethe identity of R₁₀ group and binding moiety, concentration of reactivesites on the inorganic substance, concentration of linker groups, andidentity of analyte.

In general, the concentration of R₁₀ can be in the range of about 1 toabout 10 groups per square nanometer (nm²) of support surface area,based on surface area measured by BET. In certain embodiments, thebinding moiety concentration depends primarily on the analyte sought tobe recovered when using the composition. As indicated above, theconcentration of binding moiety can also depend on the concentration ofany optional linker used. In general, however, the binding moiety can bein a concentration in the range of 0.04 to about 4 groups per squarenanometer. In addition, binding moiety is not always attached to alinker on a one to one stoichiometry. In certain embodiments, e.g., whenthe binding moiety is prepared from a large protein molecule, thebinding moiety can be attached by several linker groups. In otherembodiments employing smaller binder moieties, less than stoichiometricamounts of binding moieties are used and any unreacted linker groups are“capped” to avoid interference when the invention is used for aseparation.

The amount of R₁₀ and binding moiety can also be stated in terms of howmany functional groups on the starting inorganic substance are reactedor “covered” by the R₁₀, binding moiety, and/or or optional linker. Forexample, about 50% to about 99% of surface hydroxy groups of saidinorganic substance can be covered with the R₁₀ surface moieties andabout 1% to about 50% of the surface hydroxy groups can be covered withthe binding moiety, optionally attached to the inorganic substance viathe linker.

In certain embodiments of the solid of the present invention, about 75%to about 99% of the surface hydroxy groups of said inorganic substanceis covered with the R₁₀ surface moieties and about 1% to about 25% ofthe surface hydroxy groups is covered with the binding moiety directlyor indirectly attached to the inorganic substance via the linker.

As indicated above, the solid of this invention comprising at least onebinding moiety and R₁₀ can be employed to isolate analytes known to bindto the binding moiety. Accordingly, the present invention encompasses amethod of isolating an analyte mixed with at least one other componentin a mixture, said method comprising the following steps:

-   -   1. contacting a solid of the present invention with said        mixture, wherein the at least one binding moiety has a specific        affinity for said analyte;    -   2. allowing said analyte to bind to said at least one binding        moiety;    -   3. removing said at least one other component from the solid        said analyte bound thereto;    -   4. recovering the solid; and    -   5. isolating the analyte from the solid.

In an embodiment of the method of the present invention, the bindingmoiety is present in an amount sufficient to provide specific binding toa desired analyte. The at least one other component is removed in step(3) by washing the solid with a fluid and discarding the washate; andwherein said analyte is isolated in step (5) by placing an eluant on thesolid and collecting the eluant.

In the method of isolating the analyte, it is preferred that about 50%to about 99% of the hydroxy groups of a surface of said inorganicsubstance is covered with the surface moieties and about 1% to about 50%of the hydroxy groups of the surface is covered with the binding moietydirectly or indirectly attached to the inorganic substance via thelinker.

The method of isolating the analyte further prefers that about 75% toabout 99% of the hydroxy groups of the surface of said inorganicsubstance is covered with the surface moieties and about 1% to about 25%of the hydroxy groups of the surface is covered with the binding moietydirectly or indirectly attached to the inorganic substance via thelinker.

In a preferred embodiment of the method of isolating the analyte, saidinorganic substance is a silica gel or chromatographic grade silica.More preferably the inorganic substance is silica gel.

The present invention also includes a method of reducing nonspecificbinding of impurity (impurities are non-analyte components, i.e. speciesother than the analyte, in a mixture containing the analyte) to a solidcomprising an inorganic substance, wherein said inorganic substancecomprises at least one functional group to which non-specific bindingoccurs or which otherwise causes non-specific binding. The inorganicsubstance comprises the aforementioned inorganic oxide and the methodcomprises the following steps:

-   -   1. providing said solid;    -   2. reacting said at least one functional group with reactant to        create moiety R₁₀ on at least one surface of the inorganic        substance, wherein R₁₀ is selected from the group consisting of        —CH₂OH, —CH(OH)₂, —CH(OH)CH₃, —CH₂CH₂OH, —C(OH)₂CH₃, —CH₂CH(OH)₂        and —CH(OH)CH₂(OH).

In the method of reducing non-specific binding, R₁₀ is preferably anentity selected from the group consisting of —CH₂OH, —CH(OH)CH₃,—CH₂CH₂OH, and —CH(OH)CH₂(OH). More preferably, R₁₀ is an entityselected from the group consisting of —CH₂OH, —CH(OH)CH₃ and —CH₂CH₂OH.Most preferably, R₁₀ is —CH₂OH. R₁₀ is present on the surface of theinorganic substance in sufficient amounts such that when the inorganicsubstance is contacted with a mixture comprising impurity, non-specificbinding of the impurity to the solid is reduced.

The method is particularly useful in reducing non-specific binding toinorganic metal oxides, silicates or aluminosilicate having hydroxylfunctionality located on its surface. It is particularly useful forinorganic metal oxides such as silica (silica gel and chromatographicgrade silica), alumina, silica-alumina, zirconia, zirconate, controlledpore glass, titania, coprecipitates and mixtures thereof. The method isalso useful on magnetically responsive inorganic oxides (such assiliceous oxide-coated magnetic particles).

There are three types of binding of proteins or other species to thesupport surface that must be considered to minimize the non-selectivebinding to the solid support.

The surface charge of the support should be ideally zero at theoperating pH of the adsorption. This is due to the fact that proteinscarry a net charge due to the excess of —COOH or —NH₂ groups in theprotein. For a complex mixture of proteins at about pH 7, if a proteinin the mixture has an isoelectric point <7 it will have a net negativecharge, and conversely if a protein in the mixture has an isoelectricpoint >7 it will have a net positive charge. An unreacted silica surfacehas an isoelectric point of about 2, so if it is contacted with acomplex mixture at pH=7 or so, it will have a strong negative charge,and therefore the proteins that are positively charge will adsorbnon-selectively to the silica surface. This explains the strongnon-selective binding of proteins to silica surfaces. Therefore, asstated above, the surface charge of the support should be ideally zeroat the operating pH of the adsorption.

The second type of binding interaction that should be minimized ishydrophobic bonding. Although weaker than electrostatic or dipoleinteractions at single sites, hydrophobic interactions becomeappreciable when it becomes collective between many adjacent sites. Thehydrophobic interaction becomes dominant when the salt concentration ofthe solvent is relatively high. The ions of the salt can interact withthe charged surface, thereby “screening the charge” from the proteins.While the presence of high salt reduces the electrostatic interactionwith the surface, hydrophobic interactions become dominant, if thesurface has a hydrophobic character. Therefore, a hydrophobic surfacecomposition should be avoided on the support to minimize thisinteraction.

The third type of binding interaction of proteins to surfaces ishydrogen bonding, or a dipolar interaction. Interestingly, if thesolvent is water, this interaction will favor the solvent over thesurface due to entropy considerations. That is, if a protein has a“choice” to bind to a surface through hydrogen bonding or, remain insolution in the water system, which is also a hydrogen bondinginteraction, the solution case is favored because of its higher entropystate. Therefore, for protein purification from aqueous solution, asurface that has a dipolar composition is favored to minimizenon-selective binding. Such a surface is typically hydrophilic at lowelectrostatic charge density.

It is believed that the invention addresses each of these threeinteractions and that one of the novel features of this invention is asurface composition that presents a hydrophilic surface with very lowsurface charge density for protein purification from water basedsystems. This surface composition is achieved by chemically modifyinginorganic oxide based support, such as silica, preferably silica gelswith the aforementioned R₁₀ groups such as —CH₂OH, —CH(OH)₂, —CH(OH)CH₃,—CH₂CH₂OH, —C(OH)₂CH₃, —CH₂CH(OH)₂ and —CH(OH)CH₂(OH), preferably—CH₂OH, covering the surface. These R₁₀ groups are hydrophilic yet arevery weak acids, meaning that these R₁₀ groups are essentially notdisassociated at pH less than about 12 or so, and therefore are notcharged. When a complex mixture of proteins are presented with thissurface composition, charge interactions are minimized, yet hydrogenbonding interactions will favor the hydrogen bonding from the watersolvent due to higher entropy state of the solvated protein over aprotein hydrogen bonded to the surface. This surface composition willthen minimize non-selective binding of unwanted proteins to the highcapacity affinity support, yielding high purification factors at highcapacity.

Also in the scope of the invention are solids comprising R₁₀ moietiesand at least one linker located on a surface of the inorganic substance.Such solids can be considered an intermediate which can be sold “as is”to a user of the solid. Prior to isolating a desired analyte, the usercan then react a binding moiety to the linker group. The linker groupsmay also optionally be capped or otherwise in a precursor form whichwould require further chemistry before reacting it with a bindingmoiety.

The following working examples are presented to illustrate some of theaspects of the present invention and should not be construed to limitthe scope of the present invention. The present invention may beembodied in embodiments not illustrated by the Examples withoutdeparting from the spirit or essential attributes of the inventiondisclosed herein. For instance, the present invention can be practicedby one skilled in the art as described in the claims and any embodimentshaving elements equivalent to elements recited in the claims areincluded within the scope of the claimed invention.

EXAMPLES 1 AND 2

Non-Specific Binding on Conventional Silica Media

These examples show that the neat uncoated charged silica surface of theprior art strongly adsorbs proteins based mostly on isoelectric pointand the surface area of the silica. Two types of silicas were tested:Examples 1 and 2. Example 1 was a low surface area silica gel with asurface area=161 m²/g after 4 hours at 150° C. heat treat (micropore=73m²/g; mesopore=88 m²/g, pore volume=0.373 cc/g, average pore diameter=93Å). Example 2 was a higher surface area/pore volume silica gel, surfacearea=253 m²/g after 4 hours at 150° C. heat treat (micropore=35 m²/g;mesopore=218 m²/g, pore volume=2.445 cc/g, average pore diameter=387 Å).The examples below describe a procedure where the neat silica sampleswere contacted with a complex mixture of proteins in aqueous solution.The resultant supernatant was then analyzed by isoelectric focussing gelelectrophoresis for protein adsorption.

A vial (325 μg protein/vial) of Pharmacia 3.6-9.3 Broad pI CalibrationKit (catalog # 17-0471-01) was dissolved in 200 μl DI H₂O in aneppendorf tube. 0.005 g of Example 1 was added. In another eppendorftube, a vial (325 μg protein/vial) of Pharmacia 3.6-9.3 Broad pICalibration Kit (catalog # 17-0471-01) was dissolved in 200 μl DI H₂Oand then 0.005 g of Example 2 was added. Both samples were stirred endover end for 1 hour. These samples were run subjected to 3-9 IsoelectricFocussing Gel Electrophoresis on a Pharmacia PhastGel unit. The resultsare shown in FIG. 2. Lane Description 2, 7 Pharmacia 3.6-9.3 Broad pIStandard 3, 4 Example 1 5, 6 Example 2

FIG. 2 shows that bands (proteins) were missing from the samples thatwere contacted with Examples 1 and 2, which means that these proteinsadsorbed to the silica surfaces. The high surface area silica, Example2, adsorbed all of the proteins with isoelectric points greater than5.9, while the lower surface silica, Example 1 adsorbed only proteins ofhigher pI. The data clearly show that uncoated silica binds proteinsprimarily through a strong electrostatic interaction, and that thesurface is negatively charged at this pH (assumed to be around 5.5 orso).

EXAMPLES 3, 4, AND 5

Non-Selective Binding on Hydrophobic Treated Supports

These examples show that when the silica is coated with hydrophobicgroups, methyl or octyl groups, strong adsorption occurs, especially atmoderate ions strength of the solvent, ˜0.1 M salt. Example 3 was anuncoated neat commercial wide pore silica from W. R. Grace & Co.,XWP-gel P 005, SA=72 m²/g, with 50 nm pore median that had beenactivated for 2 hours at 150° C. Example 4 was the silica of Example 3that had been coated with methyl groups, described below. Example 5 wasthe silica of Example 3 that had been coated with octyl groups,described below. Example 4 was prepared as follows. In a 250 ml roundbottom flask, 50 ml toluene and 6.16 g of methyltriethoxysilane wereadded. Then 10.1 g of Example 3 was added to atoluene/methyltriethoxysilane solution. N₂ was flowed for 5 minutes toremove air and continued for the entire reaction. The sample wasrefluxed and stirred at 110° C. for 4 hours. The sample was thenfiltered and washed 3 times with 50 ml of toluene. The sample wasreslurried into 50 ml of toluene, then filtered and washed 3 times with50 ml of toluene. The sample was then reslurried into 50 ml of toluene,filtered and washed 3 times with 50 ml of toluene. The sample was driedat 110° C. and then calcined 4 hours at 150° C.

Example 5 was prepared as follows. 10.1 g of Example 3 was impregnatedto incipient wetness with 0.53 g of octyltriethoxysilane dissolved in13.25 g of toluene as solvent. The sample was then air-dried in a hoodfor 2 hours, dried at 110° C. for one hour and then calcined 4 hours at150° C.

Protein adsorption in 0.1 M NaCl was determined as follows. BecauseExamples 4 and 5 were hydrophobic, a wetting procedure was needed toinsure good contact with the protein solution. To an eppendorf tube,0.014 g Example 3 was added as the control. Then 1 ml ethanol was added,stirred and centrifuged with a supernatant removed. 0.5 ml ethanol and0.5 ml DI H₂O were added, stirred and centrifuged with a supernatantremoved. 0.25 ml ethanol and 0.75 ml DI H₂O were added, stirred andcentrifuged with a supernatant removed. 1 ml DI H₂O was added, stirredand centrifuged with a supernatant removed. The DI H₂O wash was repeatedfour more times. 1 ml 0.1 M NaCl+0.02 M PBS pH=7.4 were added, stirredand centrifuged with a supernatant removed. The wash with 0.1 MNaCl+0.02 M PBS pH=7.4 was repeated four more times. Two vials of SigmaIEF Mix 3.6-9.3 Isoelectric Focusing Marker (catalog # I-3018) weredissolved into 500 μl 0.1 M NaCl+0.02 M PBS pH=7.4. The dissolved IEFMix was added to an eppendorf tube.

To another eppendorf tube, 0.014 g of Example 4 was added. The samewetting procedure and protein addition as Example 3 were performed withExample 4.

To a third eppendorf tube, 0.014 g of Example 5 was added. The samewetting procedure and protein addition as Example 3 were performed withExample 5.

One vial of Sigma IEF Mix 3.6-9.3 Isoelectric Focusing Marker (catalog #I-3018) was dissolved into 250 μl 0.1 M NaCl+0.02 M PBS pH=7.4. This wasthe standard untreated protein mixture.

All samples were stirred end over end for 1 hour. The samples weresubjected to 3-9 Isoelectric Focussing Gel Electrophoresis on aPharmacia PhastGel unit. The results are shown in FIG. 3. LaneDescription 1, 8 Standard protein mixture 2, 3 Example 3 4, 5 Example 46, 7 Example 5

As seen in FIG. 3, while the surface charge of the silica was “screened”by the dissolved salt, 0.1M NaCl, and no protein binding occurred, thehydrophobic interaction of the methyl, and especially the octyl, groupswas very strong and many of the bands were missing. These data showclearly that at the conditions above, a hydrophobic surface compositioncan lead to non-selective binding.

EXAMPLES 6, 7 AND 8 EXAMPLE OF INVENTION

These Examples show the advantage of employing an R₁₀ group according tothis invention for reducing non-selective protein binding to a silicasurface. Example 6 was the same as Example 3 except it was activated 2hours at 200° C. Example 7 was an intermediate surface composition, withthe silica surface having Si—R groups attached, wherein R isacetoxymethyl. Example 8 was an example of the surface composition ofthe present invention, with the silica surface having Si—R₁₀ groupsattached, wherein R₁₀ is methylhydroxy. The advantage of Example 8 withhigh and low ionic strength solvents was also shown.

Example 7 was prepared as follows. In a 250 ml round bottom flask, 50 mltoluene and 20.42 g of acteoxymethyltriethoxysilane were added. 15.05 gof Example 6 was added to a toluene/acteoxymethyltriethoxysilanesolution. N₂ was flowed for 5 minutes to remove air and continued forthe entire reaction. The sample was refluxed and stirred at 110° C. for16 hours. Then, the sample was filtered and washed 3 times with 50 ml oftoluene. The sample was reslurried into 50 ml of toluene, filtered andwashed 5 times with 50 ml of toluene. The sample was then reslurriedinto 50 ml of toluene, filtered and washed 5 times with 50 ml oftoluene. It was dried at 110° C. then calcined 4 hours at 150° C.

The preparation of Example 8 is described as follows. In a 250 ml roundbottom flask, 10 g of Example 7 and 100 ml 0.01 M H₂SO₄ were added. N₂was flowed for 5 minutes to remove air and continued for entirereaction. The sample was refluxed and stirred at 100° C. for 18 hours.Then, the sample was filtered and washed 2 times with 100 ml 80° C. DIH₂O. The sample was reslurried into 100 ml 80° C. DI H₂O; filtered andwashed 2 times with 100 ml 80° C. DI H₂O; dried at 110° C. and thencalcined 4 hours at 150° C.

To an eppendorf tube, 0.007 g of Example 7 was added. One vial of SigmaIEF Mix 3.6-9.3 Isoelectric Focusing Marker (catalog # I-3018) wasdissolved into 250 μl 0.14 M NaCl+0.02 M PBS pH=7.2 and then added tothe eppendorf tube. The sample was labeled Example 7 high salt.

To a second eppendorf tube, 0.007 g of Example 8 was added. One vial ofSigma IEF Mix 3.6-9.3 Isoelectric Focusing Marker (catalog # I-3018) wasdissolved into 250 μl 0.14 M NaCl+0.02 M PBS pH=7.2 and then added tothe eppendorf tube. This sample was labeled Example 8 high salt.

To a third eppendorf tube, 0.007 g of Example 7 was added. One vial ofSigma IEF Mix 3.6-9.3 Isoelectric Focusing Marker (catalog # I -3018)was dissolved into 250 ul 0.02 M PBS pH=7.4 and then added to theeppendorf tube. This sample was labeled Example 7 low salt.

To a fourth eppendorf tube, 0.007 g of Example 8 was added. One vial ofSigma IEF Mix 3.6-9.3 Isoelectric Focusing Marker (catalog # I-3018) wasdissolved into 250 μl 0.02 M PBS pH=7.4 and then added to the eppendorftube. This sample was labeled Example 8 low salt.

To a fifth eppendorf tube, one vial of Sigma IEF Mix 3.6-9.3 IsoelectricFocusing Marker (catalog # I-3018) was dissolved into 250 ul DI H₂O andthen added to the eppendorf tube. This sample was labeled proteinmixture standard.

All samples were stirred end over end for 1 hour. All samples were thensubjected 3-9 Isoelectric Focussing Gel Electrophoresis on a PharmaciaPhastGel unit. The results are shown in FIG. 4. Lane Description 1, 8protein mixture standard Example 7 high salt Example 8 high salt Example7 low salt Example 8 low salt

The results of this experiment clearly show the advantage of Example 8,one of the embodiments of this invention, for rejecting nonspecificadsorption to the silica surface, in that all of the protein bands arepresent, see Lanes 3 and 7, under both “high salt” and “low salt”conditions.

Characterization of Example 8

The surface composition of this invention was characterized by analysesdescribed below.

FIG. 5 shows the diffuse reflectance infrared spectrum of Example 8,which had a surface composition comprising —CH₂OH groups, from 1400-4000cm⁻¹. The infrared data were acquired on a Nicolet Magna 550 using aSpectra-Tech diffuse reflectance accessory. The samples were diluted1:20 in KBr with 512 scans collected at 4 cm⁻¹ resolution. The peaks at2937 and 2897 cm⁻¹ clearly show the presence of the —CH₂ groups. Thebands for the —OH resonances are buried under the broad peak at 3483cm⁻¹. For comparison, FIG. 6 shows the spectrum of Example 7, with asurface composition comprising —CH₂OCOCH₃ groups. New resonancesoccurred at 1726, 1421, and 1374 cm⁻¹ which are characteristicresonances associated with the acetoxy groups.

FIG. 7 shows the MAS Si²⁹ NMR spectrum of Example 8. A single-pulse ²⁹Sinuclear magnetic resonance experiment was performed on a ChemagneticsCMX 200 operating at a resonance frequency of 39.76 MHz. The sample waspacked in a 14 mm pencil-style rotor. A pulse length of 4 μscorresponding to a 22 degree pulse was utilized along with a relaxationdelay of 60 s. The clear resonance at −62 ppm has been identified asO₃Si—CH_(x)—, see Vicic, D., and Maciel, J. Am. Chem. Soc. 105 (1983),pg. 3767-3776.

FIG. 8 shows the x-ray photoelectron spectrum of Example 8. The samplewas mounted on a sample stub with double-sided tape and a 2 hour carbon,oxygen, and silicon scan was conducted. The spectrum was fit to twopeaks which were identified as contaminant C, 284.7 eV, and a alcohol Catom, 286.7 eV, see “Handbook of X-ray Photoelectron Spectroscopy”,Moulder, J. F., Sticke, W. F., Sobol, P. E., and Bomben, K. D.,Perkin-Elmer Corp, Eden Prairie, Minn., 1992. For comparison the XPSspectrum of Example 7 is shown in FIG. 9. In this case, a peak at 289 eVassociated with the carboxyl carbon was also observed. These studiesindicate that the surface composition of Example 8, one of theembodiments of this invention, comprised methylhydroxy groups, —CH₂OH,e.g., an R₁₀ as defined herein.

The concentration of R₁₀ groups (—CH₂OH) on the product of Example 8 was2.01 groups/nm² and was calculated from the surface area of the silicasupport (72 m²/g) carbon content (1.907%) of the final product. Thesurface area was measured using conventional BET surface areamethodologies and the carbon content (% by weight) was measured using amodel C-144 LECO Carbon Analyzer.

EXAMPLE 9 Attachment of Binding Moiety and Illustration of ReducedNon-Specific Binding when Using the Invention

842 g toluene and 3.11 g 3-aminopropyltriethoxysilane were added to a2000 ml round bottom flask. Then, 200 g of Grace Davison XWP 500 Åsilica that was calcined 2 hours at 200° C. was added to the roundbottom flask, followed by the addition of 15 boiling chips. The roundbottom flask was put in a heating mantle and attached condenser. Theheating mantle was attached to the top of an orbital shaker, which wasoperated at a speed of 115 rpm. N₂ was passed through the round bottomflask and condenser to remove air during the entire reaction. The samplewas heated to boiling (˜110° C.) for 4 hours. The sample was filteredand washed with 2×200 ml toluene, dried at 115° C. and then calcined 2hours at 150° C. This sample was labeled Intermediary A. Theconcentration of the resulting —CH₃H₆NH₂ groups was calculated to be0.54 and was based on the surface area (BET) of the support (88 m²/g),carbon content (LECO) of the intermediary (0.321%) and nitrogen content(0.11%). The nitrogen content (in weight %) was determined on a CarloErba NA 1500 Analyzer and using methods based on a modified Dumasmethod, using an oxygen-containing atmosphere and thermal conductingdetection. See ASTM D5373 (for coal) and ASTM 5291.

800 ml 1 M NaCl was mixed with the Intermediary A in a beaker andstirred with a magnetic stirrer. The initial pH was 4.79 1 M HCl wasadded dropwise until pH became 2. The pH was held at 2.0 for 15 minutes.The sample was filtered and washed with 5×200 ml DI H₂O, dried at 115°C. and then calcined 2 hours at 200° C. This sample was labeledIntermediary B.

680 gr toluene and 177.25 gr acetoxymethyltriethoxysilane were mixedwith Intermediary B in a round bottom flask. 15 boiling chips were putin the round bottom flask, which was placed in a heating mantle andattached condenser. The heating mantle was attached to the top of anorbital shaker operating a speed of 115 rpm. N₂ was passed through theround bottom flask and condenser to remove air during the entirereaction. The sample was heated to boiling (˜110° C.) for 24 hours,filtered, washed with 3×200 ml toluene, dried at 115° C. and thencalcined 2 hours at 150° C. This sample was labeled Intermediary C.

900 ml dioxane and 100 ml 0.1 M H₂SO₄ were mixed with Intermediary C ina round bottom flask. 15 boiling chips were put in the round bottomflask, which was placed in a heating mantle and attached condenser. Theheating mantle was attached to the top of an orbital shaker operating ata speed of 115 rpm. N₂ was passed through the round bottom flask andcondenser to remove air for the entire reaction. The sample was heatedto boiling (˜100° C.) for 4 hours, filtered, washed with 2×200 mltoluene, dried at 115° C. and then calcined 2 hours at 150° C. Thissample was labeled Intermediary D. The concentration of R₁₀ groups(—CH₂OH) of this product was 5.65 and was measured by calculating thecarbon content of Intermediary D and then subtracting the amount ofcarbon attributable to the C₃H₆NH₂ groups, and then marking thecalculation made in Example 8. When doing so, the concentration of theC₃H₆NH₂ groups was calculated to be 0.39 and is less than thatcalculated from data on Intermediary A before conducting the chemistryto attach the R₁₀ group. Without being held to a particular theory, itis believed the slight variation in nitrogen content (0.11 vs. 0.08) isdue to standard deviation or possibly due to slight leaching of C₃H₆NH₂groups when creating the R₁₀ groups.

20.75 g Intermediary D and 400 ml coupling buffer (0.1 M Na₂PO₄+0.15 MNaCl; pH=7.0) were mixed in a 1000 ml beaker and stirred for 5 minutes.The sample was filtered to form a moist cake, which was put in a 1000 mlbeaker and then 587.66 g 50 wt. % gluteraldehyde and 5.91 g NaCNBH₃ wereadded to the beaker. The sample was stirred for 4 hours, filtered,washed with 400 ml coupling buffer and reslurried in 400 ml couplingbuffer to obtain a new sample, which was filtered, washed with 400 mlcoupling buffer and reslurried in 400 ml coupling buffer 2 more times.The re-washed and reslurried sample was filtered and then washed with400 ml coupling buffer. This sample was labeled Intermediary E.

75.44 g coupling buffer and 24.56 g Protein A from Repligen at aconcentration of 50 g Protein A per liter were added to a 250 ml roundbottom flask. 2.52 g NaCNBH₃ and Intermediary E were added to the flaskand mixed on a shaker for 4 hours. The sample was filtered and washed 4×with 100 ml coupling buffer. Then 75.44 g coupling buffer, 2.52 gNaCNBH₃ and 0.44 g ethanolamine were added to the 250 ml round bottomflask, and then mixed on a shaker for 4 hours. The sample was filteredand washed 4× with 100 ml coupling buffer. The sample was placed in 20%ethanol and stored at 4° C. This sample was labeled Example 9. From LECOCarbon, it was determined that Example 9 was 34.67 mg Protein A per gsilica.

A 0.66×2 cm I.D. affinity column packed with silica having —Si—CH₂OHdirectly attached was first equilibrated with 20 mM phosphate buffer, pH7.4. A 5 ml feed sample of 0.5 mg/ml rabbit polyclonal IgG insupernatant of Teredinobacter turnirae broth was loaded onto the column.The affinity column was then washed with the phosphate buffer until theUV absorbance at 280 nm returned to baseline. The IgG was eluted fromthe affinity column with 0.1 M acetic acid, pH3.0 at a flow rate of 1ml/min (see the narrow peak in FIG. 9). FIG. 10 shows the chromatogramof the loading, wash and elution from the affinity column by monitoringthe absorbance at 280 nm.

100 μL purified IgG from the eluent of the affinity purification shownin FIG. 10 was injected onto a size exclusion chromatography column,which was eluted with an elution buffer of 0.1M Na₂SO₄ and 0.05MNaH₂PO₄, pH 5, at a flow rate of 1 ml/min. The A₂₈₀ profile of theeluent from the size exclusion chromatography column was depicted inFIG. 11, which shows a single peak of the IgG with very little impurityfrom the cell broth. By comparison, FIG. 12 shows the size exclusionchromatogram, using the same conditions, of the starting rabbitpolyclonal IgG that was spiked into the cell broth. Based on acomparison of FIGS. 11 and 12, it was clear that the IgG purified fromthe cell broth using Example 9 (FIG. 11) was more pure than the startingIgG (FIG. 12). Thus, FIGS. 11 and 12 show that non-selective binding tothe silica media was minimized with the solid of this invention.

BIBLIOGRAPHY

-   Abercrombie, D. M. et al. Affinity Chromatography (eds. Rickwood, D.    & Hames, B. D.) (IRL Press, Washington, D.C., 1983).-   Argos, P. et al. Methods in Enzymology (ed. Deutscher, M. P.)    (Academic Press, San Deigo, Calif., 1990).-   Gagnon, P. Purification Tools for Monoclonal Antibodies (Validated    Biosystems, Tuscon, 1996).-   Hermanson, G. T., Mallia, A. K. & Smith, P. K. Immobilized Affinity    Ligand Techniques (Academic Press, Inc., San Diego, 1992).-   Mohan, S. B. et al. Affinity Separations A Practical Approach (eds.    Rickwood, D. & Hames, B. D.) (IRL Press, Oxford, 1997).-   Scopes, R. K. Protein Purification—Principles and Practice (ed.    Cantor, C. R.) (Springer, New York, 1994).-   Wheelwright, S. M. Protein Purification Design and Scale up of    Downstream Processing (John Wiley & Sons, Inc., New York, 1991).-   Wilson, R. C. et al. Protein Purification from Molecular Mechanisms    to Large-Scale Processes (ed. Comstock, M. J.) (The American    Chemical Society, Washington, D.C., 1990).-   Weetall, “Covalent Coupling Methods for Inorganic Support    Materials”, in Methods in Enzymology, vol. XLIV, edited by K.    Mosbach, pp. 134-148, 1976.

1. A solid comprising (i) inorganic substance, (ii) moiety R₁₀covalently bonded to a metal atom on at least one surface of saidinorganic substance and (iii) at least one linker, wherein saidinorganic substance is inorganic oxide, and said R₁₀ is selected from—CH₂OH, CH(OH₂), —CH(OH)CH₃, —CH₂CH₂OH, —C(OH₂)CH₃, —CH₂CH(OH₂) andCH(OH)CH₂(OH).
 2. The solid of claim 1, wherein said at least one linkeris optionally substituted bivalent chemical group.
 3. The solid of claim2, wherein the optionally substituted chemical group is hydrocarbylcomprising n —R— groups, with n being the number of —R— groups and n isan integer of at least 2, with n−1-R— groups optionally replaced with—O—, —S—, carbonyl, thiocarbonyl, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—,—OC(S, —C(S)O—, —C(S)S—, —SC(S)—, —N(R₄)—, —N(R₄)C(O)—, —C(O)N(R₄)—,—C(R₅)═N—, —N═C(R₅)—, —C(R₅)═NO—, —ON═C(R₅)—, —P—, —P(OH)O—, arylene,substituted arylene, cycloalkylene, substituted cycloalkylene,cycloalkenylene, substituted cycloalkenylene, bivalent heterocyclyl orsubstituted heterocyclyl, where R₄ and R₅ independently being H, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl,substituted aryl, aralkyl or substituted aralkyl
 4. The solid of claim1, wherein said at least one linker is bivalent optionally substitutedchemical group of about 1 to about 30 atoms in length measured from theterminus of said group to the inorganic substance, wherein the chemicalgroup comprises at least one —R— group, with said —R— group being amember selected from the group consisting of —CH₂—, —C(R₁)H—,—C(R₂)═C(R₃)— and —C≡C—, where R₁, R₂ and R₃ independently being H,alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl,substituted aryl, aralkyl or substituted aralkyl, said —R— groupoptionally replaced with —O—, —S—, carbonyl, thiocarbonyl, —OC(O)—,—C(O)O—, —SC(O), —C(O)S—, —OC(S)—, —C(S)O—, —C(S)S—, —SC(S)—, —N(R₄)—,—N(R₄)C(O)—, —C(O)N(R₄)—, —C(R₅)═N—, —N═C(R₅)—, —C(R₅)═NO—, —ON═C(R₅)—,—P—, —P(OH)O—, arylene, substituted arylene, cycloalkylene, substitutedcycloalkylene, cycloalkenylene, substituted cycloalkenylene, bivalentheterocyclyl or substituted heterocyclyl, where R₄ and R₅ independentlybeing H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl,alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl,aryl, substituted aryl, aralkyl or substituted aralkyl.
 5. The solid ofclaim 1, wherein said at least one linker is attached to the inorganicsubstance via an ether, thioether, ester, thioester, carbonate,carbamate, phosphate, phosphonate, phosphoester, phosphoramidate, amine,amide, imide, urea, thiourea, sulfonamide, sulfoxide, sulfone,disulfide, oxime, O-acyl oxime, O-carbamoyl oxime, O-acyloxyalkyl oxime,O-acyloxyalkyloxy oxime, O-oximinophosphate, O-oximinophosphonate,O-oximinophosphoramidate or C═C linkage.
 6. The solid of claim 1,wherein said at least one linker is formed from cyanogen bromide, aN-hydroxy succinimide ester, carbonyl diimidazole, reductive amination,2-fluoro-1-methyl-pyridinium toluene-4-sulfonate activation,1-ethyl-3-(3-dimethylpropyl)carbodiimide mediated amide bond formation,tosyl chloride, tresyl chloride, divinylsulfone, azlactone, cyanuricchloride, iodoacetyl or bromoacetyl activation, maleimide, pyridyldisulfide, an epoxy compound, 2-iminothiolane5,5-dithio-bis-(2-nitrobenzoic acid), hydrazide, diazonium or Mannichcondensation.
 7. The solid of claim 1, comprising about 1 to about 10R₁₀ moieties per nm² solid.
 8. The solid of claim 7, wherein saidinorganic substance is silica and R₁₀ is —CH₂OH.
 9. A method ofisolating an analyte mixed with at least one other component in amixture, said method comprising: (1) providing a solid comprising (i)inorganic substance, (ii) moiety R₁₀ (2) covalently bonded to a metalatom on at least one surface of said inorganic substance, and (iii) atleast one binding moiety capable of binding analyte, wherein said R₁₀ isselected from —CH₂OH, —CH(OH)₂, —CH(OH)CH₃, CH₂CH₂OH, —C(OH)₂CH₃,—CH₂CH(OH)₂ and —CH(OH)CH₂(OH); (2) contacting the solid with saidmixture, wherein said at least one binding moiety has a specificaffinity for said analyte; (3) allowing said analyte to bind to said atleast one binding moiety; (4) removing said at least one other componentfrom the solid having said analyte bound thereto; (5) recovering saidsolid; and (6) isolating the analyte from the solid.
 10. The method ofclaim 9, wherein said at least one other component is removed in step(4) by washing the solid with a fluid to obtain a washate and discardingthe washate; wherein said analyte is isolated in step (6) by placing aneluant on the solid and collecting the eluant.
 11. The method of claim9, wherein said solid comprises about 1 to about 10 R₁₀ moieties pernm².
 12. The method of claim 11, wherein said solid comprises about 0.04to about 4 binding moieties per nm² solid.
 13. The method of claim 9,wherein said inorganic substance is inorganic metal oxide, metalsilicate or aluminosilicate.
 14. The method of claim 13, wherein theinorganic substance is magnetically responsive.
 15. The method of claim13, wherein the inorganic metal oxide is silica, alumina,silica-alumina, zirconia, zirconate, titania, controlled pore glass ormixtures thereof.
 16. The method of claim 13, wherein the inorganicmetal oxide is chromatographic grade silica.
 17. The method of claim 13,wherein the inorganic metal oxide is a silica gel.
 18. The method ofclaim 9, wherein said inorganic metal substance is silica and R₁₀ is—CH₂OH.
 19. The method of claim 18, wherein said silica is silica gel.20. The method of claim 18, wherein said silica is chromatographic gradesilica.
 21. The method of claim 9, wherein said binding moiety is biotinand said analyte is avidin, streptavidin, a substance attached to avidinor a substance attached to streptavidin.
 22. The method of claim 9,wherein said binding moiety is avidin or streptavidin and said analyteis biotin or biotinylated.
 23. A method of reducing nonspecific bindingof impurity to a solid comprising inorganic substance, wherein theinorganic substance comprises at least one functional group to whichnon-specific binding occurs or which causes non-specific binding tooccur, further wherein said inorganic substance is inorganic oxide, andsaid method comprises: (1) providing said solid; (3) reacting the atleast one functional group of the inorganic substance with reactant tocreate moiety R₁₀ covalently banded to a metal atom on at least onesurface of the inorganic substance, wherein R₁₀ is selected from —CH₂OH,—CH(OH)₂, —CH(OH)CH₃, —CH₂CH₂OH, —C(OH)₂CH₃, —CH₂CH(OH)₂ and—CH(OH)CH₂(OH), and R₁₀ is covalently bonded to a metal atom on thesurface of said inorganic substance in sufficient amounts such that whenthe inorganic substance is contacted with a mixture comprising impurity,nonspecific binding of said impurity to said solid is reduced.
 24. Themethod of claim 23, wherein R₁₀ is attached to said inorganic substancevia a moiety or atom which is not present in the composition ofinorganic substance prior to step (2).
 25. The method of claim 23,wherein R₁₀ is an entity selected from the group consisting of —CH₂OH,—CH(OH)CH₃ and —CH₂CH₂OH.
 26. The method of claim 25, wherein R₁₀ isCH₂OH.
 27. The method of claim 23, wherein said inorganic substance isinorganic metal oxide.
 28. The method of claim 27, wherein the inorganicmetal oxide is magnetically responsive.
 29. The method of claim 27,wherein the inorganic metal oxide is silica, alumina, silica-alumina,zirconia, zirconate, titania, a controlled pore glass and the functionalgroups thereon comprise hydroxyl.
 30. The method of claim 27, whereinsaid inorganic metal oxide is chromatographic grade silica.
 31. Themethod of claim 27, wherein said inorganic metal oxide is silica gel.